Protein-protein and protein-small molecule interactions at membrane surfaces underly most signal transduction pathways and many regulatory networks. The interfacial nature of these interactions presents many challenges to understanding them in terms of molecular structures and mechanisms. One of the most informative model systems for developing this understanding is the phototransduction pathway of retinal rod outer segments, in which a light signal, photon capture by the G-protein coupled receptor (GPCR), rhodopsin, is converted into a highly amplified electrical signal, hyperpolarization of the transmembrane voltage. This proposal aims to continue work aimed at understanding the structural and mechanistic bases of two key steps: activation of the heterotrimeric G protein, transducin, by photoactivated rhodopsin (R*), through accelerated exchange of GTP for GDP on the G protein subunit (G), and activation of a cGMP-specific phosphodiesterase, PD6, by G-GTP. These reactions happen on a sub-second time scale at the surface of disk membranes, and depend critically on the environment provided by the membrane lipids, so that they must be studied in that context. The proposed work is technically innovative in that cutting-edge approaches will be developed and used to determine structures that have proven intractable to conventional crystallographic approaches, and to determine the kinetics of individual steps in the G protein activation reaction. It is conceptually innovative in that it will determine for the first time the rate-limiting step in activation of a G protein, and he first structures of a G protein regulated effectors in the presence and absence of the activated G protein. There are two specific aims: 1. to refine the structures of holo-PDE6 and its complexes with transducin and a prenyl-binding protein using cryo-electron microcopy, cryo-electron tomography, and electron crystallography, combined with fitting of high resolution structures of fragments. 2. To determine the rate- limiting step in G protein activation. The startling finding i the previous funding period that the surface density of the phototransduction G protein, transducin, can be reduced by two-thirds without effect on phototransduction kinetics, eliminates binding of G protein to photoactivated rhodopsin, R*, as a candidate for the rate-limiting step in G protein activation, and leaves open the question of which step is limiting. We will use innovative approaches including flash-activation coupled with a scintillation proximity assay and time resolved fluorescence to determine the kinetics of each sub-reaction in the activation process. Public Health Relevance Statement: Understanding the details of phototransduction can aid in understanding blinding diseases caused by disruption of this pathway. More broadly it can guide our understanding of many other G protein-coupled pathways, which are the targets of most drugs currently in use, and which regulate nearly all important physiological processes in the central nervous system and throughout the body.